The hematogenous spread of prions in transmissible spongiform encephalopathy (TSE)-infected animals has long been hypothesized, but evidence for the presence of prions in non-nervous/lymphoid tissues and blood was not available for several decades. Later studies have provided unequivocal proof of efficient TSE blood-borne infectivity [Andreoletti, O., et al., PLoS Pathog, 2012. 8(6): p. e1002782]. The knowledge that prions traffic throughout the body in blood has important implications for both human and animal health.
Variant Creutzfeldt-Jakob disease (vCJD) emerged following the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom in the 1980s and 90s. Biochemical and strain typing analysis have provided evidence indicating that vCJD originated from human exposure to BSE contaminated material. To date, 225 cases of vCJD have been diagnosed worldwide, four of which have been transmitted by non-leucodepleted blood transfusion [Team, E., Euro Surveill, 2007. 12(1): p. E070118 4; Wroe, S. J., et al., The Lancet, 2006. 368(9552): p. 2061-2067]. While leucocyte reduction has been implemented to filter prions and prion carrying cells from blood products, current filtration methodologies are unable to remove 100% of TSE infectivity [Lacroux, C., et al., PLoS One, 2012. 7(7): p. e42019.]. In addition, recent reports have revealed that 1/1,250 to 1/3,500 persons in the United Kingdom may be asymptomatic carriers of vCJD as a result of the BSE epidemic. Thus, concern exists that a secondary outbreak of vCJD may ensue involving blood-borne prion transmission originating from individuals unknowingly carrying a subclinical prion infection [McCutcheon, S., et al., PLOS ONE, 2011. 6(8): p. e23169.] The present invention addresses the need for an in vitro assay with the ability to detect the prion disease-associated isoform of prion protein (PrPD) present in whole blood.
Several animal TSEs, including chronic wasting disease (CWD) of deer and elk and hamster-adapted transmissible spongiform encephalopathy (TME) exhibit a hematogenous phase of infection, thus providing excellent TSE models for the development of an ante-mortem blood-borne PrPD detection assay.
While traditional assays, such as Western blot and immunohistochemistry (IHC), are effective for detecting large quantities of prions present in nervous and lymphoid tissue, they do not have the ability to detect the minute quantities thought to be present in bodily fluids or peripheral tissues early in infection. Rodent bioassays have the necessary sensitivity and specificity to detect hematogenous prions, but they are not realistic as rapid and cost-effective diagnostic tools. In vitro prion detection was advanced with the advent of serial protein misfolding cyclic amplification (sPMCA) [Saa, P. et al., Science, 2006. 313(5783): p. 92-4; Saa, P., et al., Methods Mol Biol, 2005. 299: p. 53-65.]. sPMCA requires less time than bioassay [Castilla, J., et al., Nat Med, 2005. 11(9): p. 982-5.], but has been hampered by a lack of consistent sensitivity and a dependence on protease digestion prior to immunoassay readout. In contrast, the real-time quaking-induced conversion (RT-QuIC) assay [Atarashi, R., et al., Nat Methods, 2007. 4(8): p. 645-50; Orrú, C. D., et al., mBio, 2011. 2(3): p. e00078-11; Wilham, J. M., et al., PLoS Pathog, 2010. 6(12): p. e1001217.] relies upon the seeded conversion of recombinant prion protein (rPrP) to PrPD and subsequent binding of thioflavin T (ThT) to the resulting amyloid isoforms [Krebs, M. R., et al., J Struct Biol, 2005. 149(1): p. 30-7.], thus offering enhanced ante-mortem prion detection and real-time fluorescence readout. However, improvement in the sensitivity and specificity of RT-QuIC would be very desirable.
The present invention provides new techniques and methodologies to improve the sensitivity and specificity of prior amplification and detection techniques, such as RT-QuIC, while maintaining fast, sensitive and consistent assays for the detection of blood-borne prions.